Stimulation of Toll-Like Receptors on T Cells

ABSTRACT

The present invention relates to compositions and methods for modulating Toll-like receptors (TLRs) for enhancing survival of activated CD4+ T cells. The enhanced survival of activated CD4+ T cells provides a means for regulating an immune response.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made, in part, using funds obtained from the U.S.Government (National Institutes of Health Grant No. AI41521), and theU.S. Government may therefore have certain rights in this invention.

BACKGROUND OF THE INVENTION

Toll-like receptors (TLRs) (Kaisho et al., 2001 Trends Immunol. 22:78)mediate the recognition of pathogen-associated molecular patterns(PAMPs) by cells of the innate immune system allowing the detection ofinfection and inflammation (Medzhitov et al., 1997, Nature 388:394). On(antigen presenting cell) APCs, PAMP engagement of TLRs promotesmaturation, a process characterized by the up-regulation of MHC andcostimulatory molecules and the secretion of proinflammatory cytokines,which in turn leads to the induction of proliferation and survivalpathways in antigen-specific CD4⁺ T cells (Kaisho et al., 2001, TrendsImmunol. 22:78). Several distinct molecular pathways contribute to theseeffects. For example, TCR engagement activates NF-κB, a transcriptionfactor that mediates many inflammatory responses and is important inmaintaining activated CD4⁺ T cell survival (Zheng et al., 2003, J. Exp.Med. 197:861). TCR survival signals are further enhanced bycostimulation through CD28 that promotes the synthesis of theprosurvival molecule BCl-x_(L) and the cytokine IL-2 (Boise et al.,1995, Immunity 3:87). IL-2 in turn provides survival signals toactivated CD4⁺ T cells through induction of Bcl-2 (Mueller et al., 1996J. Immunol. 156:1764). Furthermore, PAMP-stimulated APCs also secretetype I (interferons) IFNs and IL-15, both of which enhance activatedCD4⁺ T cell survival following cessation of APC TCR engagement (Oshiumiet al., 2003, Nat. Immunol. 4:161; Mattei et al., 2001, J. Immunol.167:1179). Thus, PAMPs clearly promote activated CD4⁺ T cell survivalindirectly by initiating maturation responses from APCs.

Interestingly, CD4⁺ T cells also express TLRs suggesting that PAMPs maydirectly induce activated CD4⁺ T cell survival (Mokuno et al., 2000, J.Immunol. 165:931; Caramalho et al., 2003, J. Exp. Med. 197:403). TLRexpression has been reported on γδT cells and regulatory CD4⁺CD25⁺ Tcells. However, the function of TLRs on CD4⁺ T cells remains poorlyunderstood. It has recently been reported that stimulation of TLR-4 onregulatory T cells increases the suppressive activity and proliferationof these cells (Caramalho et al., 2003, J. Exp. Med. 197:403). However,whether PAMPs are capable of inducing direct functional responses inactivated nonregulatory CD4⁺ T cells or whether TLR-mediated responsesin CD4⁺ T cells use the same signaling pathways that have previouslybeen described in APCs is not known.

TLR signaling is initiated through at least two pathways: one dependenton the adaptor molecule myeloid differentiation factor 88 (MyD88) and another that is MyD88 independent (Takeuchi et al., 2002, Curr. Top.Microbiol. Immunol. 270:155). All TLRs utilize the MyD88 pathway but notall TLRs are dependent on it to mediate all functional responses toPAMPs (Yamamoto et al., 2003, Science 301:640). For example,TLR-4-mediated IL-6 and TNF-αsynthesis by dendritic cells (DCs) isdependent on MyD88, but maturation responses such as costimulatorymolecule up-regulation are relatively independent (Kawai et al., 1999,Immunity 11:115; Akira et al., 2000, J. Endotoxin Res. 6:383). Incontrast, all TLR-9-mediated functional responses are dependent on MyD88(Schnare et al., 2000, Curr. Biol. 10:1139). Nevertheless, both pathwayslead to the activation of NF-κB and the mitogen-activated protein (MAP)kinases (Akira, 2003 J. Biol. Chem. 278:38105).

In view of the fact that the function of TLRs on CD4+ T cells,particularly nonregulatory CD4+ T cells, remains poorly understood, thepresent invention serves to provide insight into the role of TLRs onCD4+ T cells. In addition, many methods exist to expand and manipulatethis population of cells. However, generation of a large number of thesecells have not been successful. Thus there is a need for methods ofenhancing the survival of CD4+ T cells both in vitro and in vivo. Thepresent invention satisfies this need.

BRIEF SUMMARY OF THE INVENTION

The invention includes a composition for increasing T cell proliferationand cytokine production, wherein the composition comprises a Toll-likereceptor (TLR) ligand and a T cell stimulator.

In a preferred embodiment, the cytokine produced by the T cell is IL-2or IL-6.

In one embodiment, the invention includes a TLR ligand that is capableof activating TLR9.

In another embodiment, the invention includes a TLR ligand is capable ofactivating TLR3.

In yet another embodiment, the TLR ligand is selected from the groupconsisting of CpG DNA and poly I:C.

In a further embodiment, the TLR ligand is a combination of CpG DNA andpoly I:C.

In another embodiment, the T cell stimulator comprises an antibodyselected from the group consisting of an anti-CD3 antibody and ananti-CD28 antibody.

In a further embodiment, the T cell stimulator comprises both ananti-CD3 antibody and an anti-CD28 antibody.

The invention also includes a composition for increasing T cellproliferation and cytokine production, wherein the composition comprisesa TLR ligand, a T cell stimulator and an antigen having at least oneepitope, wherein the epitope is capable of eliciting an immune responsein a mammal.

The invention also includes a composition for increasing T cellproliferation and cytokine production, wherein the composition comprisesa TLR ligand, a T cell stimulator and a T cell.

In one embodiment, the T cell is an activated T cell. Preferably, the Tcell exhibits an enhanced survival characteristic.

The invention also includes a composition comprising one of CpG and polyI:C and a T cell stimulator.

In one embodiment, the T cell stimulator comprises an antibody selectedfrom the group consisting of an anti-CD3 antibody and an anti-CD28antibody.

In another embodiment, the T cell stimulator comprises both an anti-CD3antibody and an anti-CD28 antibody.

The invention also includes a T cell that is genetically modified toexpress elevated levels TLR3 and/or TLR9 compared to an otherwiseidentical T cell not so modified, wherein contact of TLR3 and/or TLR9with a TLR ligand enhances the survival of said genetically modified Tcell.

In one embodiment, the genetically modified T cell exhibits an enhancedsurvival characteristic compared to an otherwise identical T cell not somodified. Preferably, the T cell is capable of regulating an immuneresponse.

In a further embodiment, the immune response is associated with adisease selected from the group consisting of an infectious disease, acancer, and an autoimmune disease.

The invention also includes a method of inducing T cell proliferationand promoting cytokine production, the method comprising activating a Tcell with a composition comprising a TLR ligand and a T cell stimulator.

In one embodiment, the T cell proliferation is dependent on NF-κB.

In another embodiment, the method of inducing T cell proliferation andpromoting cytokine production is independent of the presence of anantigen presenting cell.

The invention also includes a method of inducing T cell proliferationand promoting cytokine production, the method comprising activating a Tcell with a composition comprising one of CpG and poly I:C; and a T cellstimulator.

In one embodiment, the T cell stimulator comprises an antibody selectedfrom the group consisting of an anti-CD3 antibody and an anti-CD28antibody.

In another embodiment, the T cell stimulator comprises both an anti-CD3antibody and an anti-CD28 antibody.

In another embodiment, the method of inducing T cell proliferation andpromoting cytokine production is independent of the presence of anantigen presenting cell.

In another embodiment, the cytokine is selected from the groupconsisting of IL-2 and IL-6.

The invention also includes a method of enhancing an immune response ina mammal, the method comprising administering to the mammal acomposition comprising a TLR ligand a T cell stimulator.

The invention further provides a method of enhancing an immune responsein a mammal, the method comprising administering to the mammal acomposition comprising one of CpG and poly I:C; and a T cell stimulator.

The invention also includes a method of enhancing an immune response ina mammal, the method comprising administering to the mammal a T cellthat has been stimulated with a composition comprising a TLR ligand anda T cell stimulator.

The invention also provides a method of enhancing an immune response ina mammal, the method comprising administering to the mammal a T cellthat has been stimulated with a composition comprising one of CpG andpoly I:C; and a T cell stimulator.

Also included in the invention is a method of suppressing an immuneresponse in a mammal, the method comprising administering to the mammala composition that inhibits and/or reduces expression of a TLR and/or adownstream signaling molecule thereof in a T cell in the mammal.Preferably, the composition is selected from the group consisting of asmall interfering RNA (siRNA), an antisense nucleic acid and a ribozyme.

In addition, the invention includes a method of suppressing an immuneresponse in a mammal, the method comprising administering to the mammala composition that inhibits and/or reduces activity of a TLR and/or adownstream signalling molecule thereof in a T cell in the mammal.Preferably, the composition is selected from the group consisting of atransdominant negative mutant, an intracellular antibody, a peptide anda small molecule.

The invention also includes a method of modulating Foxp3 expression in Tcells, the method comprising activating a T cell with a compositioncomprising one of CpG and poly I:C and a T cell stimulator wherein saidstimulator is capable of activating said T cell.

In one embodiment, the composition comprises CpG and Foxp3 expression isreduced.

In another embodiment, the composition comprises poly I:C and Foxp3expression is induced.

In one aspect, the composition further comprises transforming growthfactor beta (TGF-b).

The invention is also directed to the use of a composition comprising aToll-like receptor (TLR) ligand and a T cell stimulator for preparationof a medicament for: a method of inducing T cell proliferation andpromoting cytokine production; a method of enhancing an immune responsein a mammal; and a method of modulating Foxp3 expression in a T cell.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 is a chart depicting TLR RNA expression patterns in activatedCD4⁺CD25⁻ T cells.

FIG. 2 is a chart demonstrating that Poly(I:C) or CpG DNA but not LPSinduce NF-κB and MAPK signaling in activated CD4⁺ T cells.

FIG. 3, comprising FIGS. 3A through 3D, is a series of chartsdemonstrating that Poly(I:C) or CpG DNA directly enhances the survivalbut not the proliferation of activated CD4⁺ T cells.

FIG. 4, comprising FIGS. 4A through 4D, is a series of chartsdemonstrating that Poly(I:C) or CpG DNA-mediated survival requires NF-κBactivation and is associated with Bcl-x_(L) up-regulation but only CpGDNA-mediated survival is MyD88 dependent.

FIG. 5, comprising FIGS. 5A and 5B, is a chart demonstrating thatactivated CD4⁺ T cell survival in vivo is enhanced by either poly(I:C)or CpG DNA treatment before adoptive transfer into naive hosts.

FIG. 6 is a chart demonstrating that resting mouse CD4⁺CD25⁻ T cellsexpress TLR9 protein, whereas CD4⁺CD25⁺ Tregs do not.

FIG. 7, comprising FIGS. 7A through 7D, is a series of chartsdemonstrating that Poly I:C or CpG DNA is able to synergize with T cellstimulation to induce T cell proliferation.

FIG. 8 is a chart demonstrating that Poly I:C or CpG DNA is able tosynergize with T cell stimulation in IL-2 protein production.

FIG. 9 is a chart demonstrating that Poly I:C and CpG DNA mediatedproliferative responses are TRAF 6 independent.

FIG. 10, comprising FIGS. 10A and 10B, is a series of chartsdemonstrating CpG DNA stimulated Akt phosphorylation and GSKαphosphorylation in a PI3-kinase dependent manner in CD4⁺ T cells.

FIG. 11 is a chart demonstrating that CpG DNA mediated IL-2 synthesis inCD4⁺ T cells is MyD88 and PI3-kinase dependent.

FIG. 12, comprising FIGS. 12A and 12B, is a series of chartsdemonstrating that CpG DNA mediated enhancement of CD4⁺ T cellproliferation is MyD88 dependent.

FIG. 13 is a chart demonstrating that MyD88 has a highly conservedputative SH2 binding (YXXM) domain. Majority-SEQ ID NO:17. Human MyD88TIR-SEQ ID NO:18. Murine MyD88 TIR-SEQ ID NO:19. Zebrafish MyD88 TIR-SEQID NO:20.

FIG. 14 is a schematic depicting an experimental model with respect to achimera with MyD88-deficient T cells to assess the role of MyD88 in Tcell responses in vivo.

FIG. 15 is a chart demonstrating that chimeric mice with MyD88-deficientT cells have splenocytes that upregulated CD86 expression in thepresence of CpG DNA.

FIG. 16 is a chart demonstrating that chimeric mice with MyD88-deficientT cells have less plasma INF-γ (INF-g) and IL-12 after infection with Tgondii.

FIG. 17, comprising FIGS. 17A and 17B, is a schematic depicting a signaltransduction pathway involving MyD88 (FIG. 17A). FIG. 17B depicts astrategy for retroviral reconstitution of MyD88−/− CD4⁺ T cells.

FIG. 18 is a chart demonstrating that optimal IL-6 response to LPS orIL-1 is dependent on Y257 residue in a putative SH2 binding sequence inthe MyD88 TIR domain.

FIG. 19, comprising FIGS. 19A through 19D, is a series of chartsdemonstrating that the death domain and residue Y257 of the TIR domainof MyD88 are both required for optimal CpG ODN-induced proliferation ofCD4+ T cells.

FIG. 20 is a chart demonstrating that chimeric mice with MyD88-deficientT cells have similar survival to MyD88−/− mice in that both fail tosurvive the acute phase T. gondii infection.

FIG. 21, comprising FIGS. 21A and 21B, is a series of graphsdemonstrating that TLR ligands can modify Foxp3 expression in naturalTregs.

FIG. 22 is a series of graphs demonstrating the effect of TLR ligands onTGF-b induction of Foxp3 expression in adaptive Tregs.

FIG. 23, comprising FIGS. 23A and 23B, is a series of two chartsdemonstrating that CpG, but not poly I:C, induces IL-6 production inboth Th cells and Tregs.

DETAILED DESCRIPTION

The invention relates to the discovery that activated CD4⁺ T cells orotherwise pre-stimulated T cells express Toll-like receptor (TLR)-3 andTLR-9 but not TLR-2 and TLR-4, and that the treatment of activated CD4⁺T cells with ligands for TLR-3 and/or TLR-9 promotes T cell survival. Insome cases, the T cell survival was observed without augmentingproliferation of the T cell. In addition, the invention relates to thediscovery that activation of a TLR on a T cell at the time of T cellstimulation induces a heightened rate of cellular proliferation andpromotes enhanced cytokine production. As such, the present inventionencompasses compositions and methods for activating a TLR on a T cellprior to, concurrently with, or following stimulation of the T cell.

The present invention includes compositions and methods for activatingToll-like receptors (TLRs) on T cells to induce multiple signallingpathways, to promote T cell proliferation and survival and to promotethe development of effector T cell function, including, but not limitedto, development of memory T cells. The invention also includescompositions and methods for manipulating TLRs on T cells to modulate animmune response. The invention also includes compositions and methodsfor modulating Foxp3 expression in T cells. In addition, the presentinvention includes compositions and methods that can be used to developactive vaccines and adoptive immunotherapy.

The invention is applicable in systems where T cells are expanded exvivo by stimulation with antibodies to CD3 and/or CD28 in the absence ofAPCs. However, the invention should not be limited to anti-CD3 andanti-CD28 antibodies for stimulating T cells, but rather any stimulatorof T cells can be used. The stimulation of T cells can be additive whena TLR is activated using the methods disclosed herein, such as usingagents including, but not limited to, CpG DNA and poly I:C to enhancethe survival characteristics of the T cells.

The invention also provides a method of manipulating T cell activationin ex vivo cultures that is not dependent upon the presence of APCs. Anapplication of the present invention includes the areas of immuneadjuvants (for vaccines and cancer immunotherapy).

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used.

“Activation”, as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“An antigen presenting cell” (APC) is a cell that is capable ofactivating T cells, and includes, but is not limited to,monocytes/macrophages, B cells and dendritic cells (DCs).

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia areata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillian-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

The term “DNA” as used herein is defined as deoxyribonucleic acid.

“Donor antigen” refers to an antigen expressed by the donor tissue to betransplanted into the recipient.

“Recipient antigen” refers to a target for the immune response to thedonor antigen.

As used herein, an “effector cell” refers to a cell which mediates animmune response against an antigen. Effector cells include, but are notlimited to, T cells and B cells.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “enhanced survival characteristic,” refers to the discoverythat following contacting a TLR ligand with a corresponding TLR on a Tcell, levels of prosurvival molecules such as BCl-X_(L) are up-regulatedcompared with a T cell not so contacted.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

The term “expression vector” as used herein refers to a vectorcontaining a nucleic acid sequence coding for at least part of a geneproduct capable of being transcribed. In some cases, RNA molecules arethen translated into a protein, polypeptide, or peptide. In other cases,these sequences are not translated, for example, in the production ofantisense molecules, siRNA, ribozymes, and the like. Expression vectorscan contain a variety of control sequences, which refer to nucleic acidsequences necessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well.

The term “heterologous” as used herein is defined as DNA or RNAsequences or proteins that are derived from the different species.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC5′ and 3′TATGGC5′ share 50%homology.

As used herein, “homology” is used synonymously with “identity.”

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, i.e., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, i.e., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, i.e., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (i.e.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

As used herein, the term “modulate” is meant to refer to any change inbiological state, i.e. increasing, decreasing, and the like.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

The term “polypeptide” as used herein is defined as a chain of aminoacid residues, usually having a defined sequence. As used herein theterm polypeptide is mutually inclusive of the terms “peptide” and“protein”.

“Proliferation” is used herein to refer to the reproduction ormultiplication of similar forms of entities, for example, proliferationof a cell. That is, proliferation encompasses production of a greaternumber of cells, and can be measured by, among other things, simplycounting the numbers of cells, measuring incorporation of ³H-thymidineinto the cell, and the like.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a cell substantiallyonly if the cell is a cell of the tissue type corresponding to thepromoter.

The term “RNA” as used herein is defined as ribonucleic acid.

The term “recombinant DNA” as used herein is defined as DNA produced byjoining pieces of DNA from different sources.

The term “recombinant polypeptide” as used herein is defined as apolypeptide produced by using recombinant DNA methods.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally-occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are culture in vitro. In other embodiments, the cells are notcultured in vitro.

By the term “specifically binds,” as used herein, is meant an antibody,or a ligand, which recognizes and binds with a cognate binding partner(e.g., a stimulatory and/or costimulatory molecule present on a T cell)protein present in a sample, but which antibody or ligand does notsubstantially recognize or bind other molecules in the sample.

The term “T-cell,” as used herein, is defined as a thymus-derived cellthat participates in a variety of cell-mediated immune reactions.

As used herein, a “T cell stimulator,” means an antibody and/or a ligandthat, when specifically bound with a cognate binding partner on a Tcell, mediates a response by the T cell, including, but not limited to,activation, initiation of an immune response, proliferation, cytokineproduction and the like. A T cell stimulator can include, but is notlimited to, an MHC molecule loaded with a peptide, an anti-CD3 antibody,an anti-CD28 antibody, an antigen and the like.

As used herein, a “therapeutically effective amount” is the amount of atherapeutic composition sufficient to provide a beneficial effect to amammal to which the composition is administered.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

The term “vaccine” as used herein is defined as a material used toprovoke an immune response after administration of the material to amammal.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

The term “virus” as used herein is defined as a particle consisting ofnucleic acid (RNA or DNA) enclosed in a protein coat, with or without anouter lipid envelope, which is capable of replicating within a wholecell.

DESCRIPTION

The invention relates to the identification of a novel mechanism bywhich T cells respond to engagement of TLRs with their respectiveligand. The disclosure presented herein demonstrate that activated Tcells express TLR-3 and TLR-9 but not TLR-2 and TLR-4. Activation ofTLR-3 and/or TLR-9 on T cells directly enhance their survival in a NF-κBdependent manner demonstrating that TLRs on T cells can directlymodulate the immune response. In addition, the invention relates to thediscovery that activation of a TLR on a T cell at the time of T cellstimulation induces a heightened rate of cellular proliferation andcytokine production.

Based on the present disclosure, T cell development, including but notlimited to, proliferation and survival, can be regulated by manipulatinga TLR on a T cell. As such, the present invention includes compositionsand methods for modulating the expression and/or activity of TLRs on a Tcell to regulate survival and proliferation of the cell. The compositionof the present invention is useful in providing a therapeutic benefit incell therapy and/or vaccination.

In an embodiment of the invention, a T cell in which a TLR has beenactivated exhibits an enhanced survival characteristic compared with anotherwise identical T cell not having the TLR activated. Preferably, theTLR activated is TLR-3 and/or TLR-9.

According to the present invention, a T cell can be expanded in vitro bycontacting a TLR with the appropriate TLR ligand on the T cell at thetime of T cell stimulation. That is, the invention relates to thediscovery that activation of a TLR on a T cell at the time of T cellstimulation induces a heightened rate of cellular proliferation andcytokine production. Preferably, the cytokine is IL-2. In any event,following treatment and culturing of the T cells in vitro according tothe methods disclosed herein the T cells are immunologically functional.For example, they are capable of inducing an immune response andtherefore can be administered to a patient in need thereof.

In addition to enhancing T cell survival and cytokine production byactivating a TLR on the T cell and stimulating the T cell with a T cellstimulator, the present invention also includes compositions and methodsfor negatively regulating T cell activation. As such, the inventionencompasses compositions and methods for suppressing an immune response.As more fully discussed elsewhere herein, one such method is to decreasethe expression or inactivate the protein involved in the TLR signalingpathway including, but not limited to, the TLR itself and downstreamsignaling molecules. One skilled in the art will appreciate, based onthe disclosure provided herein, that one way to decrease the mRNA and/orprotein levels of a TLR and/or a downstream signaling molecule in a Tcell is by reducing or inhibiting expression of the nucleic acidencoding the TLR and/or the downstream signaling molecule. Thus, theprotein level of the TLR and/or the downstream signaling molecule in theT cell can also be decreased using a molecule or compound that inhibitsor reduces gene expression such as, for example, an antisense molecule,an siRNA or a ribozyme. Alternatively, the activation of the TLR and/orthe downstream signaling molecule can be reduced or inhibited by atransdominant negative mutant of the TLR and/or the downstream signalingmolecule.

Regulation of Toll-Like Receptor (TLR)

Based on the disclosure herein, the present invention includes thegeneric concept for modulating TLR expression and/or activity in a cell.Preferably, the TLR is TLR3 and/or TLR-9. However, the invention shouldnot be construed to only encompass TLR3 and TLR9, but rather include anyTLR that is found to induce proliferation of T cells when contacted withits corresponding ligand. Generating a T cell that exhibits an increasedexpression and/or activity of a TLR provides a means to promote cellularsurvival and proliferation. As discussed elsewhere herein, cellularsurvival refers to the fact that following activation of a TLR on a Tcell, various signal transduction molecules are activated, such as, butnot limited to, BCl-X_(L), Akt, NF-κB, MyD88, and the like.

With respect to TLR3, it has been demonstrated that activation of TLR3on a T cell promotes T cell survival. Preferably, activation of the TLR3with poly I:C induces activation of the T cell in a MyD88-independentmanner.

With respect to TLR9, it has been demonstrated that activation of TLR9on a T cell promotes T cell survival. Preferably, activation of the TLR9with CpG DNA induces activation of the T cell in a MyD88-dependentmanner.

However, while it is thought that the effects of CpG described hereinresult solely from CpG interaction with TLR9, it is possible that CpGinteraction with another TLR or a non-TLR mediated receptor may alsocontribute to the observed effects of CpG.

Based on the present disclosure activation of TLR3 and/or TLR9 on a Tcell induces survival of the T cell without affecting its innate abilityto modulate the immune response. Thus, manipulation of a TLR on a Tcell, for example activating expression and/or activity of a TLR on a Tcell, offers a strategy to induce T cell proliferation and survivalthereby inducing an immune response. In addition, activation of a TLRsuch as TLR3 and/or TLR9 on a T cell promotes cytokine production.Preferably, the cytokine is IL-2.

Expression of a TLR, preferably TLR3 and/or TLR9 can be induced in acell using a composition comprising an expression vector encoding theTLR. One skilled in the art will appreciate, based on the disclosureprovided herein, that one way to increase the mRNA and/or protein levelsof a TLR in a cell is by inducing expression of a nucleic acid encodingthe desired TLR.

Based on the present disclosure, one skilled in the art will recognizethat in addition to being able to activate a T cell and thereby induce aT cell response with respect to activating a TLR on the T cell, thepresent invention also includes compositions and methods for suppressinga T cell response. In view of the fact that TLRs contribute to survivalcharacteristics and cytokine production in T cells, it can beappreciated that the effects of TLRs on T cell survival can be reducedor inhibited. Such a method can involve decreasing the expression orinactivate the protein involved in the TLR signaling pathway including,but not limited to the TLR itself, and downstream signaling molecules(i.e. PI3-kinase, Akt, GSKα, NF-κB, MyD88 and others). One skilled inthe art will appreciate, based on the disclosure provided herein, thatone way to decrease the mRNA and/or protein levels of a TLR in a cell isby reducing or inhibiting expression of the nucleic acid encoding theTLR. Thus, the protein level of the TLR and downstream signalingmolecules in a cell can also be decreased using a molecule or compoundthat inhibits or reduces gene expression such as, for example, anantisense molecule, an siRNA or a ribozyme.

An siRNA is an RNA molecule comprising a set of nucleotides that istargeted to a gene or polynucleotide of interest. As used herein, theterm “siRNA” encompasses all forms of siRNA including, but not limitedto (i) a double stranded RNA polynucleotide, (ii) a single strandedpolynucleotide, and (iii) a polynucleotide of either (i) or (ii) whereinsuch a polynucleotide, has one, two, three, four or more nucleotidealterations or substitutions therein.

Based on the present disclosure, it should be appreciated that thesiRNAs of the present invention may effect the target polypeptideexpression to different degrees. The siRNAs thus must first be testedfor their effectiveness. Selection of siRNAs are made therefrom based onthe ability of a given siRNA to interfere with or modulate theexpression of the target polypeptide.

In yet another embodiment, the expression of the desired TLR and/or thedownstream signaling molecule can be inhibited using an antisensenucleic acid sequence. Preferably, the antisense nucleic acid isexpressed by a plasmid vector. The antisense expressing vector is usedto transfect a mammalian cell or the mammal itself, thereby causingreduced endogenous expression of the desired TLR and/or the downstreamsignaling molecule in the cell. However, the invention should not beconstrued to be limited to inhibiting expression of the desired TLRand/or the downstream signaling molecule by transfection of cells withantisense molecules. Rather, the invention encompasses other methodsknown in the art for inhibiting expression or activity of a protein inthe cell including, but not limited to, the use of a ribozyme.

Ribozymes and their use for inhibiting gene expression are also wellknown in the art (see, e.g., Cech et al., 1992, J. Biol. Chem.267:17479-17482; Hampel et al., 1989, Biochemistry 28:4929-4933;Eckstein et al., International Publication No. WO 92/07065; Altman etal., U.S. Pat. No. 5,168,053). Ribozymes are RNA molecules possessingthe ability to specifically cleave other single-stranded RNA in a manneranalogous to DNA restriction endonucleases. Through the modification ofnucleotide sequences encoding these RNAs, molecules can be engineered torecognize specific nucleotide sequences in an RNA molecule and cleave it(Cech, 1988, J. Amer. Med. Assn. 260:3030). A major advantage of thisapproach is the fact that ribozymes are sequence-specific.

There are two basic types of ribozymes, namely, tetrahymena-type(Hasselhoff, 1988, Nature 334:585) and hammerhead-type. Tetrahymena-typeribozymes recognize sequences which are four bases in length, whilehammerhead-type ribozymes recognize base sequences 11-18 bases inlength. The longer the sequence, the greater the likelihood that thesequence will occur exclusively in the target mRNA species.Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating specific mRNA species, and18-base recognition sequences are preferable to shorter recognitionsequences which may occur randomly within various unrelated mRNAmolecules.

In another aspect of the invention, the desired TLR and/or thedownstream signaling molecule can be inhibited by way of inactivatingand/or sequestering the protein. As such, inhibiting the effects of aTLR and/or a downstream signaling molecule can be accomplished by usinga transdominant negative mutant. Alternatively an intracellular antibodyspecific for the desired protein may be used. In one embodiment, theantagonist per se is a protein and/or compound having the desirableproperty of interacting with a binding partner of the TLR and/or thedownstream signaling molecule and thereby competing with thecorresponding wild-type protein. In another embodiment, the antagonistis a protein and/or compound having the desirable property ofinteracting with the TLR and/or the downstream signaling molecule andthereby sequestering the protein. In any event, the TLR and/or thedownstream signaling molecule is inhibited and thereby reducing orpreventing the normal outcome of activating a TLR and/or a downstreamsignaling molecule in a T cell.

One skilled in the art will readily appreciate that as a result of thedegeneracy of the genetic code, many different nucleotide sequences mayencode the same polypeptide. That is, an amino acid may be encoded byone of several different codons, and a person skilled in the art canreadily determine that while one particular nucleotide sequence maydiffer from another, the polynucleotides may in fact encode polypeptideswith identical amino acid sequences. As such, polynucleotides that varydue to differences in codon usage are specifically contemplated by thepresent invention for the purpose of regulating expression and/oractivity of a TLR.

Vectors

Whether the purpose is to increase expression or inhibit expression of amRNA and/or protein level of a desired TLR and/or a downstream signalingmolecule, the invention includes an isolated nucleic acid encoding a TLRand/or a downstream signaling molecule, operably linked to a nucleicacid comprising a promoter/regulatory sequence such that the nucleicacid is preferably capable of directing expression of the proteinencoded by the nucleic acid. In other related aspects, the inventionincludes an isolated nucleic acid encoding a TLR and/or a downstreamsignaling molecule.

The invention encompasses expression vectors and methods for theintroduction of exogenous DNA into cells with concomitant expression ofthe exogenous DNA in the cells. The incorporation of a desiredpolynucleotide into a vector and the choice of vectors is well-known inthe art as described in, for example, Sambrook et al. (2001, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York),and in Ausubel et al. (1997, Current Protocols in Molecular Biology,John Wiley & Sons, New York).

The polynucleotide of the invention can be cloned into a variety ofvectors. However, the present invention should not be construed to belimited to any particular vector. Instead, the present invention shouldbe construed to encompass a wide plethora of vectors which are readilyavailable and/or well-known in the art. For example, an thepolynucleotide of the invention can be cloned into a vector including,but not limited to a plasmid, a phagemid, a phage derivative, a mammalvirus, and a cosmid. Vectors of particular interest include expressionvectors, replication vectors, probe generation vectors, and sequencingvectors.

In specific embodiments, the expression vector is selected from thegroup consisting of a viral vector, a bacterial vector and a mammaliancell vector. Numerous expression vector systems exist that comprise atleast a part or all of the compositions discussed above. Prokaryote-and/or eukaryote-vector based systems can be employed for use with thepresent invention to produce polynucleotides, or their cognatepolypeptides. Many such systems are commercially and widely available.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001), and in Ausubel et al.(1997), and in other virology and molecular biology manuals. Viruses,which are useful as vectors include, but are not limited to,retroviruses, adenoviruses, adeno-associated viruses, herpes viruses,and lentiviruses. In general, a suitable vector contains an origin ofreplication functional in at least one organism, a promoter sequence,convenient restriction endonuclease sites, and one or more selectablemarkers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193.

For expression of the TLR and/or the downstream signaling molecule, atleast one module in each promoter functions to position the start sitefor RNA synthesis. The best known example of this is the TATA box, butin some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 genes, a discrete element overlying the start site itselfhelps to fix the place of initiation.

Additional promoter elements, i.e., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either co-operativelyor independently to activate transcription.

A promoter may be one naturally associated with a gene or polynucleotidesequence, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment and/or exon. Such a promoter canbe referred to as “endogenous.” Similarly, an enhancer may be onenaturally associated with a polynucleotide sequence, located eitherdownstream or upstream of that sequence. Alternatively, certainadvantages will be gained by positioning the coding polynucleotidesegment under the control of a recombinant or heterologous promoter,which refers to a promoter that is not normally associated with apolynucleotide sequence in its natural environment. A recombinant orheterologous enhancer refers also to an enhancer not normally associatedwith a polynucleotide sequence in its natural environment. Suchpromoters or enhancers may include promoters or enhancers of othergenes, and promoters or enhancers isolated from any other prokaryotic,viral, or eukaryotic cell, and promoters or enhancers not “naturallyoccurring,” i.e., containing different elements of differenttranscriptional regulatory regions, and/or mutations that alterexpression. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (U.S. Pat. No.4,683,202, U.S. Pat. No. 5,928,906). Furthermore, it is contemplated thecontrol sequences that direct transcription and/or expression ofsequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the celltype, organelle, and organism chosen for expression. Those of skill inthe art of molecular biology generally know how to use promoters,enhancers, and cell type combinations for protein expression, forexample, see Sambrook et al. (2001). The promoters employed may beconstitutive, tissue-specific, inducible, and/or useful under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous.

Constitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV40) early promoter, immediate earlycytomegalovirus (CMV) promoter sequence, mouse mammary tumor virus(MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR)promoter, Moloney virus promoter, the avian leukemia virus promoter,Epstein-Barr virus immediate early promoter, Rous sarcoma viruspromoter, as well as human gene promoters such as, but not limited to,the actin promoter, the myosin promoter, the hemoglobin promoter, andthe muscle creatine promoter. Further, the invention should not belimited to the use of constitutive promoters. Inducible promoters arealso contemplated as part of the invention. The use of an induciblepromoter in the invention provides a molecular switch capable of turningon expression of the polynucleotide sequence which it is operativelylinked when such expression is desired, or turning off the expressionwhen expression is not desired. Examples of inducible promoters include,but are not limited to a metallothionine promoter, a glucocorticoidpromoter, a progesterone promoter, and a tetracycline promoter. Further,the invention includes the use of a tissue specific promoter, whichpromoter is active only in a desired tissue. Tissue specific promotersare well known in the art and include, but are not limited to, the HER-2promoter and the PSA associated promoter sequences.

In order to assess the expression of a TLR and/or a downstream signalingmolecule, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other embodiments, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers are known in the art and include, for example,antibiotic-resistance genes, such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Reportergenes that encode for easily assayable proteins are well known in theart. In general, a reporter gene is a gene that is not present in orexpressed by the recipient organism or tissue and that encodes a proteinwhose expression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells.

Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (see, e.g.,Ui-Tei et al., 2000 FEBS Lett. 479:79-82). Suitable expression systemsare well known and may be prepared using well known techniques orobtained commercially. Internal deletion constructs may be generatedusing unique internal restriction sites or by partial digestion ofnon-unique restriction sites. Constructs may then be transfected intocells that display high levels of siRNA polynucleotide and/orpolypeptide expression. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast or insectcell by any method in the art. For example, the expression vector can betransferred into a host cell by physical, chemical or biological means.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York), and in Ausubel et al. (1997, Current Protocols in MolecularBiology, John Wiley & Sons, New York).

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Apreferred colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (i.e., an artificial membrane vesicle). Thepreparation and use of such systems is well known in the art.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the polynucleotide of thepresent invention, in order to confirm the presence of the recombinantDNA sequence in the host cell, a variety of assays may be performed.Such assays include, for example, “molecular biological” assays wellknown to those of skill in the art, such as Southern and Northernblotting, RT-PCR and PCR; “biochemical” assays, such as detecting thepresence or absence of a particular peptide, e.g., by immunologicalmeans (ELISAs and Western blots) or by assays described herein toidentify agents falling within the scope of the invention.

In the case where a non-viral delivery system is utilized, a preferreddelivery vehicle is a liposome. The above-mentioned delivery systems andprotocols therefore can be found in Gene Targeting Protocols, 2ed., pp1-35 (2002) and Gene Transfer and Expression Protocols, Vol. 7, Murrayed., pp 81-89 (1991).

T Cell Stimulator

A T cell stimulator of the present invention includes an antibody and/ora ligand that when specifically bound with a cognate binding partner ona T cell, mediates a response by the T cell, including, but not limitedto, activation, initiation of an immune response, proliferation,cytokine production, and the like. A T cell stimulator can include, butis not limited to, an MHC molecule loaded with a peptide (otherwiseknown as peptide/MHC tetramer), an anti-CD3 antibody, an anti-CD28antibody, an antigen and the like.

The present invention includes various methods for stimulating a T cellincluding, but not limited to, contacting a T cell with whole antigen inthe form of a protein, cDNA or mRNA. However, the invention should notbe construed to be limited to the specific form of the antigen used forstimulating the T cell. Rather, the invention encompasses other methodsknown in the art for generating stimulated T cell. Preferably, the Tcell is contacted with an anti-CD3 antibody. In another aspect, the Tcell is contacted with an anti-CD28 antibody. In yet another aspect, theT cell is contacted with both an anti-CD3 antibody and an anti-CD28antibody. As discussed elsewhere herein, the T cell can be stimulatedprior to, concurrently with, or following activation of a TLR on the Tcell.

The invention includes a T cell that has been exposed or otherwise“activated” with a T cell stimulator and activated by the T cellstimulator. For example, a T cell can be activated by contacting with aT cell stimulator before, after or concurrently with contacting TLR withits corresponding ligand on the T cell. A result of such a treatment isthe generation of an activated cell exhibiting an enhanced survivalcharacteristic and enhanced cytokine production. In the case where anantigen is used to activate the T cell, the result is anantigen-specific T cell exhibiting an enhanced survival signal andenhanced cytokine production. The T cell may become activated in vitro,e.g., by culture ex vivo in the presence of an antigen, or in vivo byexposure to an antigen.

A skilled artisan would also readily understand that a T cell can be“activated” in a manner that exposes the T cell to a T cell stimulatorfor a time sufficient to promote activation of signal transductionpathways indicative of T cell activation. For example, a T cell can beexposed to an antigen in a form small peptide fragments, known asantigenic peptides.

The antigen-specific T cell of the invention is produced by exposure ofthe T cell to an antigen either in vitro or in vivo. In the case wherethe T cell is contacted with an antigen in vitro, the T cell is platedon a culture dish and exposed to an antigen in a sufficient amount andfor a sufficient period of time to allow the antigen to bind to the Tcell and induce T cell activation. The amount and time necessary toachieve binding of the antigen to the T cell may be determined by usingmethods known in the art or otherwise disclosed herein. Other methodsknown to those of skill in the art, for example immunoassays or bindingassays, may be used to detect the presence of antigen on the T cellfollowing exposure to the antigen.

The antigen may be derived from a virus, a fungus, or a bacterium. Theantigen may be a self-antigen or an antigen associated with a diseaseselected from the group consisting of an infectious disease, a cancer,an autoimmune disease.

It is understood that an antigenic composition of the present inventionmay be made by a method that is well known in the art, including but notlimited to chemical synthesis by solid phase synthesis and purificationaway from the other products of the chemical reactions by HPLC, orproduction by the expression of a nucleic acid sequence (e.g., a DNAsequence) encoding a peptide or polypeptide comprising an antigen of thepresent invention in an in vitro translation system or in a living cell.In addition, an antigenic composition can comprise a cellular componentisolated from a biological sample. Preferably the antigenic compositionis isolated and extensively dialyzed to remove one or more undesiredsmall molecular weight molecules and/or lyophilized for more readyformulation into a desired vehicle. It is further understood thatadditional amino acids, mutations, chemical modification and such like,if any, that are made in a antigen component will preferably notsubstantially interfere with the antibody recognition of the epitopicsequence.

Methods

The invention encompasses a method for inducing proliferation of a Tcell. In another embodiment, the invention includes a method forexpanding a population of T cells. The T cell so induced or expandedexhibits an enhanced survival characteristic and enhanced cytokineproduction following treatment of the T cell according to the methodsdisclosed herein. The method comprises contacting a T cell that is to beexpanded with a TLR ligand and a T cell stimulator. As demonstratedelsewhere herein, contacting a T cell with a TLR ligand and a T cellstimulator, stimulates the T cell and induces T cell proliferation suchthat large numbers of T cells can be readily produced. In the event thatan antigen-specific T cell is desired, an antigen can be contacted witha T cell before, concurrently with or after activating a TLR on the Tcell. The T cell can be further purified using a wide variety of cellseparation and purification techniques, such as those known in the artand/or described elsewhere herein.

The invention encompasses a method for inducing a T cell response to anantigen in a mammal. The method comprises administering a compositioncomprising a TLR ligand and a T cell stimulator that specificallyinduces proliferation of a T cell specific for the antigen and inducesproduction of a cytokine. Once sufficient numbers of antigen-specific Tcells are obtained using the TLR ligand and T cell stimulator to expandthe T cell, the antigen-specific T cells so obtained are administered tothe mammal according to the methods disclosed elsewhere herein, therebyinducing a T cell response to the antigen in the mammal. This isbecause, as demonstrated by the data disclosed herein, thatantigen-specific T cells can be readily produced by stimulating restingT cells using the compositions of the invention.

The invention encompasses a method for modulating Foxp3 expression in Tcells. Foxp3 expression is a hallmark of regulatory T cells (Tregs). Itis thought that Foxp3 functions as a Treg cell lineage specificationfactor, and is necessary and sufficient for regulatory function in Tcells. Modulating Foxp3 expression permits the modulation of the numberof Tregs in a T cell population. The number of Tregs can be increased byinducing Foxp3 expression or the number can be decreased by reducingFoxp3 expression. Modulating the number of Tregs may be useful intherapeutic applications. The method comprises activating a T cell witha composition comprising a Toll-like receptor (TLR) ligand and a T cellstimulator. In one embodiment, the TLR ligand is CpG and Foxp3expression is reduced. In another embodiment, the TLR ligand is poly I:Cand Foxp3 expression is induced. The composition for inducing Foxp3expression optionally further comprises transforming growth factor beta(TGF-b).

Therapeutic Application

In one embodiment, the invention includes a vaccine. Preferably, thevaccine is a cellular vaccine, whereby a cell may be isolated from aculture, tissue, organ or organism and administered to a mammal in needthereof. The cell may also express one or more additional vaccinecomponents, such as immunomodulators or adjuvants. In a preferredembodiment, the cellular vaccine of the present invention comprises a Tcell exhibiting an enhanced survival characteristic and enhancedcytokine production compared to an otherwise identical T cell nottreated using the methods of the present invention. The T cellcomprising the vaccine has been contacted with a composition comprisinga TLR ligand and a T cell stimulator.

In another embodiment, the cellular vaccine comprises a T cell that hasbeen manipulated according to the present invention to acquire increasedexpression and/or activity of a TLR (i.e. TLR3 and/or TLR9) and/or adownstream signaling molecule. The T cell can also be cultured in vitroin the presence of both a TLR ligand and a T cell stimulator to expandthe number of T cells sufficient for therapeutic and/or experimentaluse. A benefit of generating a T cell that has been activated bycontacting with a TLR ligand and a T cell stimulator is that suchtreatment does not perturb the capacity of the cells to modulate theimmune response. Preferably, the treatment of the T cells does notperturb the capacity of the cells to suppress a disease in vivo. Yet thetreatment of the cells with a TLR ligand and a T cell stimulator allowsfor the rapid expansion of T cells. Based on the disclosure herein, Tcells treated according to the methods of the present invention exhibita enhanced survival characteristic. In addition, the T cells followingtreatment with a TLR ligand and a T cell stimulator exhibit an enhancedcytokine production. Preferably, the T cell exhibits enhanced IL-2expression.

Following the treatment and culturing of the T cells in vitro, the cellscan be administered to a patient in need thereof. Ex vivo procedures arewell known in the art and are discussed more fully below. Briefly, cellsare isolated from a mammal (preferably a human) and activated in vitro.The cells can also be genetically modified (i.e., transduced ortransfected in vitro) with a vector expressing a polynucleotide of thepresent invention. In any event, the cell can then be administered to amammalian recipient to provide a therapeutic benefit. The mammalianrecipient may be a human and the cell so modified can be autologous withrespect to the recipient. Alternatively, the cells can be allogeneic orsyngeneic with respect to the recipient.

With respect to administering a T cell of the present invention to apatient in need thereof, a T cell can be manipulated to exhibit anenhanced survival characteristic as well as increased cytokineproduction using the methods of the present invention. Based on thepresent invention, TLR activation on a T cell increases the survivalcharacteristic and cytokine production of the cell, but does not perturbthe biological function of the T cell. For example, treatment of a Tcell according to the present invention does not perturb the capacity ofthe T cell to induce an immune response in vivo. With respect tomanipulation of a T cell to induce expression of a TLR, it is envisionedthat such a T cell exhibits an increased survival characteristic andcytokine production following activation of the TLR and stimulation ofthe T cell with a stimulatory of the present invention.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells.

The T cells expanded according to the present invention are administeredto a mammal. The amount of cells administered can range from about 1million cells to about 300 billion. The cells may be infused into themammal or may be administered by other parenteral means. The mammal ispreferably a human patient. The precise dosage administered will varydepending upon any number of factors, including but not limited to, thetype of mammal and type of disease state being treated, the age of themammal and the route of administration.

The cell may be administered to a mammal as frequently as several timesdaily, or it may be administered less frequently, such as once a day,once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the mammal, etc.

A T cell (or cells expanded thereof) may be co-administered to themammal with the various other compounds (cytokines, chemotherapeuticand/or antiviral drugs, among many others). Alternatively, thecompound(s) may be administered an hour, a day, a week, a month, or evenmore, in advance of the T cell (or cells expanded thereby), or anypermutation thereof. Further, the compound(s) may be administered anhour, a day, a week, or even more, after administration of T cell (orcells expanded thereby), or any permutation thereof. The frequency andadministration regimen will be readily apparent to the skilled artisanand will depend upon any number of factors such as those alreadydiscussed elsewhere herein.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to regulate an immune response in amammal.

With respect to in vivo immunization, the present invention provides ause of a composition for increasing T cell proliferation wherein thecomposition comprises a TLR ligand and/or a T cell stimulator. As such,a vaccine useful for in vivo immunization comprises at least a TLRligand and/or a T cell stimulator component. In another aspect, thevaccine further comprises an antigen component, wherein the antigencomponent is capable of eliciting an immune response in a mammal.

The invention encompasses in vivo immunization for cancer and infectiousdiseases. In one embodiment, the disorder or disease can be treated byin vivo administration of a TLR ligand and/or a T cell stimulator aloneor in combination with an antigen to generate an immune response againstthe antigen in the patient. Based on the present disclosure,administration of a TLR ligand and/or a T cell stimulator in combinationwith a antigenic formulation enhances the potency of an otherwiseidentical vaccination protocol without the use of a TLR ligand and/or aT cell stimulator. Without wishing to be bound by any particular theory,it is believed that immune response to the antigen in the patientdepends upon (1) the composition comprising a TLR ligand and/or a T cellstimulator administered, (2) the duration, dose and frequency ofadministration, (3) the general condition of the patient, and ifappropriate (4) the antigenic composition administered.

In one embodiment, the mammal has a type of cancer which expresses atumor-specific antigen. In accordance with the present invention, animmunostimulatory protein can be made which comprises a tumor-specificantigen sequence component. In such cases, the TLR ligand and/or a Tcell stimulator is administered in combination with an immunostimulatoryprotein to a patient in need thereof, resulting in an improvedtherapeutic outcome for the patient, evidenced by, e.g., a slowing ordiminution of the growth of cancer cells or a solid tumor whichexpresses the tumor-specific antigen, or a reduction in the total numberof cancer cells or total tumor burden.

In a related embodiment, the patient has been diagnosed as having aviral, bacterial, fungal or other type of infection, which is associatedwith the expression of a particular antigen, e.g., a viral antigen. Inaccordance with the present invention, an immunostimulatory protein maybe made which comprises a sequence component consisting of the antigen,e.g., an HIV-specific antigen. In such cases, a composition comprising aTLR ligand and/or a T cell stimulator is administered in combinationwith the immunostimulatory protein to the patient in need thereof,resulting in an improved therapeutic outcome for the patient asevidenced by a slowing in the growth of the causative infectious agentwithin the patient and/or a decrease in, or elimination of, detectablesymptoms typically associated with the particular infectious disease.

In either situation, the disorder or disease can be treated byadministration of a TLR ligand and/or a T cell stimulator in combinationwith an antigen to a patient in need thereof. The present inventionprovides a means to generate a T cell induced immune response to theantigen in the patient. Based on the present disclosure, a skilledartisan would appreciate that a proinflammatory cytokine (i.e. IL-12,TNFα, IFNα, IFNβ, IFNγ and the like) can be added to the treatmentregiment disclosed herein to enhance the potency of the compositioncomprising a TLR ligand and/or a T cell stimulator.

The invention also encompasses the use of pharmaceutical compositions ofan appropriate protein or peptide and/or isolated nucleic acid topractice the methods of the invention.

The pharmaceutical compositions useful for practicing the invention maybe administered to deliver a dose of between 1 ng/kg/day and 100mg/kg/day. In one embodiment, the invention envisions administration ofa dose which results in a concentration of the compound of the presentinvention between 1 μM and 10 μM in a mammal.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the description of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as non-human primates, cattle, pigs, horses,sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, or another route of administration. Othercontemplated formulations include projected nanoparticles, liposomalpreparations, resealed erythrocytes containing the active ingredient,and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers and AZT, protease inhibitors, reverse transcriptaseinhibitors, interleukin-2, interferons, cytokines, and the like.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Remington's PharmaceuticalSciences (1985, Genaro, ed., Mack Publishing Co., Easton, Pa.), which isincorporated herein by reference.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations which are evident as a result of the teachings providedherein.

Example 1 Toll-Like Receptor Ligands Directly Promote Activated CD4⁺ TCell Survival

TLR engagement by pathogen-associated molecular patterns (PAMPs) is animportant mechanism for optimal cellular immune responses. APC TLRengagement indirectly enhances activated CD4⁺ T cell proliferation,differentiation, and survival by promoting the up-regulation ofcostimulatory molecules and the secretion of proinflammatory cytokines.However, TLRs are also expressed on CD4⁺ T cells, indicating that PAMPsmay also act directly on activated CD4⁺ T cells to mediate functionalresponses. The results disclosed herein demonstrate that activated mouseCD4⁺ T cells express TLR-3 and TLR-9 but not TLR-2 and TLR-4. Treatmentof highly purified activated CD4⁺ T cells with the dsRNA syntheticanalog poly(I:C) and CpG oligodeoxynucleotides (CpG DNA), respectiveligands for TLR-3 and TLR-9, directly enhanced their survival withoutaugmenting proliferation. In contrast, peptidoglycan and LPS, respectiveligands for TLR-2 and TLR-4 had no effect. Enhanced survival mediated byeither poly(I:C) or CpG DNA required NF-κB activation and was associatedwith Bcl-x_(L) up-regulation. However, CpG DNA, but notpoly(I:C)-mediated effects on activated CD4⁺ T cells required theTLR/IL-1R domain containing adaptor molecule myeloid differentiationfactor 88 (MyD88). Collectively, the results disclosed hereindemonstrate that PAMPs can directly promote activated CD4⁺ T cellsurvival, demonstrating that TLRs on T cells can directly modulateadaptive immune responses.

The materials and methods employed in the experiments disclosed hereinare now described.

Mice

BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor,Me.). DO11.10 mice on the BALB/c background have been describedpreviously (Hsieh et al., 1992, Proc. Natl. Acad. Sci. USA 89:6065).MyD88^(−/−) mice have been described previously (Adachi et al., 1998,Immunity 9:143). For these experiments MyD88^(+/−) mice were backcrossedat least five times onto a C57BL/6 background and intercrossed togenerate MyD88^(−/−) and MyD88^(+/+) wild-type control littermates.

CD4⁺ T Cell Purification

In experiments using BALB/c CD4⁺ T cells, splenocytes and lymph nodecells were pooled, erythrocyte-depleted by hypotonic lysis, and labeledwith CD4-FITC monoclonal antibody (GK1.5; BD Biosciences, Mountain View,Calif.) and CD25-PE monoclonal antibody (PC61; BD Biosciences). Labeledcells were sorted by a FACSVantage high-speed sorter (BD Biosciences)into CD25⁻CD4⁺ populations and then incubated with CD44-biotinmonoclonal antibody (IM7; BD Biosciences) and the following mixture ofbiotinylated monoclonal antibody from the MACS CD4⁺ T cell isolation kit(Miltenyi Biotec, Auburn, Calif.): CD8a (Ly-2), CD11b (Mac-1), CD45R(B220), pan NK (DX5), and Ly-76 (TER-119). These cells were then furtherincubated with anti-biotin magnetic beads (Miltenyi Biotec) and purifiedover LS columns (Miltenyi Biotec) in accordance with the manufacturer'srecommendations to obtain the naive CD44^(low) CD25⁻CD4⁺ T cell fraction(purity>99%). In experiments using DO11.10, MyD88^(−/−) or MyD88^(+/+)wild-type littermate control mice, CD4⁺ T cells were directly purifiedfrom erythrocyte-depleted splenocyte and lymph node cells with the MACSCD4⁺ T cell isolation kit. Purity in these fractions exceeded 96%. Theremainder of the cells were CD8⁺ T cells. APC contamination could not bedetected by FACS analysis. However, the presence of APCs was routinelyassessed by RT-PCR for MHC class II IAβ message. By the limits ofdetection in the RT-PCR assay, exceeding one APC in 1000 CD4⁺ T cells,the purified CD4⁺ T cell preparations used in all of these studies have<0.1% APC contamination.

CD4⁺ T Cell Activation

All CD4⁺ T cell activation was conducted in complete culture mediumcomposed of RPMI 1640 (Life Technologies, Grand Island, N.Y.), 1.5 μM2-ME (Sigma-Aldrich, St. Louis, Mo.), 50 μg/ml gentamicin (LifeTechnologies), and 10% FCS (Mediatech, Washington, D.C.) at 37° C. in 5%CO₂. Purified CD4⁺ T cells from either BALB/c, MyD88^(−/−), orMyD88^(+/+) wild-type control littermates were activated on 24-wellplates (Costar, Cambridge, Mass.) coated with 1.0 μg/ml CD3ε monoclonalantibody (2C11; BD Biosciences) and 1.0 μg/ml CD28 monoclonal antibody(37.51; BD Biosciences) for 16 hours. In experiments with DO11.10 mice,1 μg/ml of pOVA, a peptide derived from chicken albumin amino acidresidues 322-332 was added to 2×10⁶/ml erythrocyte-depleted splenocyteand lymph node cell pools for 16 hours. Following pOVA-inducedactivation, CD4⁺ T cell APC complexes were disrupted with 5 mM EDTA/PBSfor 10 minutes at 25° C., washed twice in PBS, and purified withmagnetic beads using the MACS CD4⁺ T cell isolation kit as describedelsewhere herein. Purity of activated DO11.10 CD4⁺ T cells exceeded 96%.As described elsewhere herein, the remainder of the contaminants by FACSanalysis were CD8⁺ T cells, with APCs at <0.1% by RT-PCR.

Semiquantitative RT-PCR

APCs were prepared from BALB/c pooled splenocyte and lymph node cellsthat were T cell depleted with MACS anti-CD90.2 beads (Miltenyi Biotec).APC, naïve, and activated CD4⁺ T cell total RNA was prepared by lysiswith RLT buffer (Qiagen, Valencia, Calif.) and with buffers and columnssupplied from the RNAeasykit with DNase I (Qiagen) in accordance withthe manufacturer's instructions. RNA was then reversed transcribed usingand amplified with the TITANIUM One Step RT-PCR kit (ClontechLaboratories, Palo Alto, Calif.) under nonsaturating conditions. Thefollowing PCR cycling conditions were used: one cycle at 95° C. for 3min followed by 25-28 cycles of 94.5° C. for 30 seconds and 60° C. for 1minute and a final cycle at 72° C. for 20 minute. Specific primersequences were as follows: 5′ TLR-2, TGCATCACCGGTCAGAAAACAACT (SEQ IDNO:1); 3′ TLR-2, GGCCCGAACCAGGAGGAAGATAAA (SEQ ID NO:2); 5′ TLR-3,CCCCTCGCTCTTTTTATGGAC (SEQ ID NO:3); 3′ TLR-3, CCTGGCCGCTGAGTTTTTGTTC(SEQ ID NO:4); 5′ TLR-4, GCCCCGCTTTCACCTCTG (SEQ ID NO:5); 3′ TLR-4,TGCCGTTTCTTGTTCTTCCTCT (SEQ ID NO:6); 5′ TLR-5, CAGCCCCGTGTTGGTAATA (SEQID NO:7); 3′ TLR-5, CCCGGAATGAAGAATGGAG (SEQ ID NO:8); 5′ TLR-9,CTATACAGCCTGCGCGTT-CTCTTC (SEQ ID NO:9); 3′ TLR-9,AGCTTGCGCAGGCGGGTTAGGTTC (SEQ ID NO:10); 5′I-Aβ^(d),ACGC-GGGCCGAGGTGGACA (SEQ ID NO:11); 3′ I-Aβ^(d),GCCCCCGATGCGGGCTCAAC (SEQ ID NO:12); 5′ G3PDH, ACCACAGTCCATGCCATCAC (SEQID NO:13); and 3′ G3PDH, TCCACCACCCTGTTGCTGTA (SEQ ID NO:14). PCRproducts were resolved by 2% agarose gel electrophoresis, stained withethidium bromide, and imaged with a Gel Doc analyzer (Bio-Rad, Hercules,Calif.).

TLR Ligand and Inhibitor Reagents

The CpG oligonucleotide TCCATGACGTTCCTGACGTT (SEQ ID NO:15) (CpG DNA)and non-CpG oligonucleotide TCCATGAGCTTCCTGAGCTT (SEQ ID NO:16) (non-CpGDNA) have been described previously (Hemmi et al., 2000, Nature 408:740)and were synthesized on a phosphorothioate backbone and purified by HPLC(Life Technologies). Poly(I:C), poly(C), and poly(dI:dC) were purchasedfrom Amersham Biosciences (Arlington Heights, Ill.) and LPS, derivedfrom the O55:B5 Escherichia coli strain, was purchased fromSigma-Aldrich. PGN was purchased from Invitrogen (Carlsbad, Calif.). TLRligands used in all experiments were dissolved in PBS except for PGN,which was solubilized in PBS with 0.02% ethanol. SB203580, U0126,NEMO-binding domain peptide (NBD), and NBD-C were all dissolved in DMSOand purchased from Calbiochem (La Jolla, Calif.).

NF-κB and MAP Kinase Signaling Analysis

BALB/c CD44^(low)CD25⁻CD4⁺ T cells were activated with plate-bound 1.0μg/ml anti-CD3 and 1.0 μg/ml anti-CD28 monoclonal antibodies for 16hours, washed, and rested for 8 hours at 37° C. Activated (1.5×10⁶) CD4⁺T cells were then treated with TLR ligands for the indicated times,lysed in 1×SDS loading buffer (Bio-Rad Life Sciences), resolved on a 12%bis-Tris SDS-PAGE gel (Life Technologies), transferred to nitrocellulosefilters (Life Technologies), and either probed with rabbit anti-mousephospho-specific Abs for p-IκBα, p-p38, p-extracellular signal-regulatedkinase (ERK) 1/2, or p-C-Jun N-terminal kinase (JNK)/stress-activatedprotein kinase (SAPK). To assess total amounts of signaling molecules,filters were also probed with either rabbit anti-mouse IκBα, p-38,ERK-1/2, or JNK/SAPK antibodies. Detection was conducted withHRP-conjugated goat ant-rabbit antibodies, ECL reagent (Amersham), andX-OMAT Film (Kodak, Rochester, N.Y.). All antibodies were purchased fromCell Signal Technologies (Beverly, Mass.).

Survival and Proliferation Analysis

Following activation, purified CD4⁺ T cells were washed twice in PBS andreplated in culture medium at 10⁶/ml. Cultures were left untreated ortreated with either TLR ligands and/or inhibitors for indicated timesand concentrations. Following incubation, CD4⁺ T cells were washed twicein PBS/2% FBS and stained with CD4-allophycocyanin monoclonal antibodyand 7-aminoactinomycin D (7-AAD; BD PharMingen) or annexin (BDPharMingen) and survival was assessed by exclusion of either of thesetwo stains. For absolute live cell counts 50,000 CD45.1⁺ splenocyteslabeled with anti-CD45.1-PE monoclonal antibody (A20; BD PharMingen)were also added to stained CD4⁺ T cell sample FACS tubes just beforeFACS analysis. Live CD4⁺ T cell counts were calculated by taking theratio of the number of CD4⁺CD45.1⁻7-ADD⁻ events collected to the numberCD45.1⁺ events collected and multiplying by 50,000. Proliferation wasmeasured by CFSE dye dilution as previously described (Wells et al.,1997 J. Clin. Invest. 100:3173).

Adoptive Transfer and Ex Vivo Proliferation Analysis

Five million purified DO11.10 CD4⁺ T cells were activated by pOVA-pulsedAPCs (1 μg/ml), purified with magnetic beads, treated with poly(I:C) (90μg/ml), CpG DNA (30 μM), LPS (100 ng/ml), or left untreated for 16hours, washed in PBS twice, and then adoptively transferred into BALB/chosts. At day 30, spleen and peripheral lymph nodes were harvested andstained with anti-CD4 monoclonal antibody and the DO11.10 clonotypicmonoclonal antibody KJI-26. Survival of activated DO11.10 CD4⁺ T cellsin each host was determined by FACS analysis and is expressed as apercent with respect to the total number of CD4⁺ T cells found in eitherthe host spleen or lymph nodes along with a mean (thick line) for eachtreatment group. To measure ex vivo proliferative responses, 72-hourquadruplicate cultures were prepared in 96-well plates with 50,000irradiated T cell-depleted pOVA-pulsed (1 μg/ml) splenocytes and 150,000CD4⁺ T cells purified with the MACS CD4⁺ T cell isolation kit from day30 peripheral lymph nodes or spleen pooled from each treatment group.[³H]Thymidine was added to cultures for an additional 8 hour and read ona beta scintillation counter and is expressed as a mean for eachtreatment group ±SEM.

Analysis of Antiapoptotic Molecules

For Bcl-2 evaluation, CD4⁺ T cells were permeabilized with 0.1%saponin/0.2% FBS/PBS and stained with anti-Bcl-2 PE monoclonal antibody(3F-11; BD PharMingen) or an isotype hamster IgG-PE control (A19-3; BDPharMingen) and staining was analyzed by FACS. Bcl-x_(L), Bcl-3, andβ-actin were analyzed by Western blotting using rabbit anti-mouse Absspecific for BCl-x_(L) (BD Transduction Laboratories, Lexington, Ky.),Bcl-3 (Santa Cruz Biotechnology, Santa Cruz, Calif.), and β-actin(Accurate Labs, Westbury, N.Y.).

The results of the experiments are now described.

CD4⁺ T Cells Modulate TLR Expression in Response to TCR Stimulation

To examine TLR expression patterns in activated CD4⁺ T cells, highlypurified naive CD44^(low)CD25⁻CD4⁺ T cells were either left to rest for8 hours or activated for 16 hours with plate-bound anti-CD3 andanti-CD28 monoclonal antibodies and RT-PCR was performed undernonsaturating conditions for TLRs that are known to have naturallyoccurring ligands (FIG. 1). TLR-2, -3, -4, -5, and -9 expression wasdetected in naive CD4⁺ T cells before activation. However, afterstimulation, TLR-4 and TLR-2 RNA expression was undetectable while TLR-3and TLR-9 message was up-regulated. To exclude the possibility that thispattern of TLR expression was partially a result of APC contamination,I-Aβ chain expression was also examined. By the limits of detection ofthis RT-PCR measurement, which is sensitive to <0.1% APC contamination,no observable I-Aβ chain RNA expression was detected in CD4⁺ T cellsbefore or after activation. Therefore the results disclosed hereindemonstrate that CD4⁺ T cells express TLR RNA and modulate itsexpression following TCR stimulation.

Poly(I:C) and CpG DNA but not LPS Induce NF-κB and MAP Kinase Activityin Activated CD4⁺ T Cells

TLR ligands induce the activation of nuclear factor NF-κB and MAPkinases in APCs (Akira, 2003, J. Biol. Chem. 278:38105; Martin et al.,2002 Biochem. Biophys. Acta. 1592:265). The next set of experiments weredesigned to assess whether TLR ligands could also induce NF-κB and MAPkinase activity in activated CD4⁺ T cells (FIG. 2). Both the TLR-3ligand poly(I:C) and the TLR-9 ligand CpG DNA were able to induce rapidNF-κB activity as evident by phosphorylation of IκBα. In a likewisemanner, both ligands were also able to effect phosphorylation of p38 MAPkinase (MAPK), ERK 1/2, and JNK/SAPK. In contrast, LPS did not inducedetectable IκBα or MAP family kinase activity consistent with theabsence of TLR-4 in activated CD4⁺ T cells. Thus, TLR ligands are ableto activate downstream signaling pathways in a manner concordant withthe cognate TLR expression pattern in activated CD4⁺ T cells.

Poly(I:C) or CpG DNA Directly Enhance Activated CD4⁺ T Cell Survival

TLR ligands directly promote the survival of several cell typesincluding neutrophils, DC, and B cells (Lundqvist et al., 2002, CancerImmunol. Immunother. 51:139; Sabroe et al., 2003, J. Immunol. 170:5268;Grillot et al., 1996, J. Exp. Med. 183:381; Grillot et al., 1996, J.Exp. Med. 183:381). Since it was observed that activated CD4⁺ T cellsalso express TLRs and signal in response to TLR ligands, the next set ofexperiments were designed to assess whether TLR ligands could directlyenhance survival of these cells. DO11.10 CD4⁺ T cells, that encode atransgenic TCR specific for a peptide derived from chicken OVA (pOVA),were activated with pOVA-pulsed APCs for 16 hours ex vivo, purified bymagnetic beads to remove all non-CD4⁺ T cells, and replated inunsupplemented culture medium in the absence or presence of TLR ligands.Survival was then assessed 72 hours following activation (FIG. 3A).Poly(I:C) or CpG DNA induced increases of activated CD4⁺ T cell survivalfrom 38% to 71 and 73%, respectively. By contrast, PGN or LPS did notsignificantly enhance activated CD4⁺ T cell survival promoting onlymarginal increases from 38% to 41% and 43%, respectively. TLR ligandenhanced mediated survival was comparable to IFN-α, which has beenpreviously reported to enhance the survival for activated T cells(Marrack et al., 1999, J. Exp. Med. 189:521), improving survival from38% to 67%. It was also observed activated CD4⁺ T cell survival inpoly(I:C) and CpG DNA-treated cultures in a dose-dependent manner (FIG.3B). Enhanced survival was not a nonspecific response to nucleic acidssince the addition poly(C), poly(dI:dC), and a control non-CpG DNA hadno significant effect. Moreover, LPS and PGN treatment also did notproduce significant increments in survival consistent with the absenceof detectable TLR-2 and TLR-4 RNA expression on activated CD4⁺ T cells.

To exclude the possibility that poly(I:C) or CpG DNA enhanced survivalwas an indirect effect arising from contamination with APCs or othernon-T cell TLR-bearing cells in the purified CD4⁺ T cell preparations,TLR ligand-treated cultures were also spiked with a T cell-depletedpreparation of pooled lymph node cells and splenocytes (APC). Theaddition of APCs to poly(I:C) and CpG DNA-treated cultures significantlyincreased dose-responsive survival over TLR ligand-treated culturesalone, suggesting synergy between indirect and direct mechanisms ofactivated CD4⁺ T cell survival. However, in LPS— or PGN-treatedcultures, it was observed that only the addition of APCs could enhancethe survival of activated CD4⁺ T cells, thus indicating the functionalabsence of other TLR-responsive cells in our purified CD4⁺ T cellpreparations.

Since percentage survival measurements may not be reflective of viableCD4⁺ T cell numbers in vitro, absolute live cell number counts werequantified for periods up to 72 hours following activation of FACSsorted CD4⁺ T cells with plate-bound anti-CD3 plus anti-CD28 monoclonalantibodies (FIGS. 3C). Consistent with increases in percentage cellsurvival, concordant increases in live CD4⁺ T cell numbers were observedin poly(I:C) or CpG DNA-treated cultures. By comparison, there were nosignificant increases in live cell numbers in LPS— or PGN-treatedcultures relative to untreated cultures.

Without wishing to be bound by any particular theory, the increases inlive cells numbers observed could be explained by TLR ligand-mediatedincreases in CD4⁺ T cell proliferation. In B cells, LPS, CpG DNA, andpoly(I:C) can induce proliferation independently of the B cell receptor(Andersson et al., 1972, Eur. J. Immunol. 2:349; Sun et al., 1997, J.Immunol. 159:3119; Alexopoulou et al., 2001, Nature 413:732). Moreover,LPS has been recently shown to augment regulatory CD4⁺ T cellproliferation (Caramalho et al., 2003, J. Exp. Med. 197:403). To addressthis issue, CFSE-labeled DO11.10 CD4⁺ T cells were first activated bypOVA-pulsed APCs for 16 hours, purified by magnetic beads, and thentreated with TLR ligands and assessed for proliferation 72 hoursfollowing activation (FIG. 3D). As expected LPS and PGN treatment, whichdid not promote direct enhancement of survival of activated CD4⁺ Tcells, did not induce more robust proliferative responses in comparisonto untreated activated controls. However, and unlike the cytokine IL-2,poly(I:C) and CpG DNA also did not enhance proliferation relative tountreated controls, indicating that the observed increases in viableCD4⁺ T cell numbers were not due to proliferation differences acrosscultures but solely reflected the enhancement of cell survival.

Poly(I:C) and CpG DNA-Mediated Survival of Activated CD4⁺ T Cells isDependent on NF-κB Activation

Activation of NF-κB is known to be associated with survival responses inactivated CD4⁺ T cells (Zheng et al., 2003, J. Exp. Med. 197:861);Hildeman et al., 2002, Curr. Opin. Immunol. 14:354). To determinewhether TLR ligand augmented survival responses in activated CD4⁺ Tcells was also dependent on NF-κB activation, IκB phosphorylation wasinhibited by using a lipid-soluble peptide (NBD) that selectively bindsto the NF-κB essential modifier (NEMO) and blocks its association withthe IκB kinases IKKα and IKKβ (IKKαβ) (May et al., 2000 Science289:1550) (FIG. 4A). NEMO-IKKαβ interaction is necessary for IκBsignal-induced phosphorylation and thus inhibiting this associationprevents subsequent IκB degradation and NF-κB translocation to thenucleus (Yamaoka et al., 1998, Cell 93:1231). It has been shown that NBDdoes not modulate JNK activity unlike peptides that directly inhibitNF-κB translocation (May et al., 2000, Science 289:1550). The resultspresented herein demonstrate that blockade of NF-κB activation by NBDinhibited the ability of both poly(I:C) or CpG DNA to enhance activatedCD4⁺ T cell survival. These effects were dose dependent. For example, at20 μM NBD, TLR ligand augmentation of activated CD4⁺ T cell wassubstantially reversed. As a control, cultures were treated with aclosely related but inactive lipid-soluble form of the peptide NBD-C andobserved no significant loss of TLR ligand-mediated survival.

MAPK p38 and ERK 1/2 are also activated by TLR ligands and theirfunction has been shown to be important in controlling T cell-mediatedinflammatory responses including survival (Schafer et al., 1999, J.Immunol. 162:659). Therefore, the next set of experiments were designedto assess whether MAPK p38 or ERK 1/2 activation is necessary for TLRligand-mediated survival in activated CD4⁺ T cells. The ERK1/2activation inhibitor U0126 or the MAPK p38 inhibitor SB203580 was addedto TLR ligand-treated activated CD4⁺ T cells and viability was assessed.It was observed that neither U0126 or SB203580 treatment decreasedpoly(I:C) and CpG DNA enhanced survival although it was observed that aconcentration of 10 μM U0126 induced a small increase in overallsurvival rates of TLR-treated activated CD4⁺ T cells. Thus, NF-κB butnot MAPK p38 or ERK1/2 activation is required to mediate TLRligand-induced survival of activated CD4⁺ T cells.

CpG DNA but not poly(I:C)-Mediated Survival of Activated CD4⁺ T Cells isDependent on MyD88

MyD88 is an adaptor molecule recruited to TLRs by TLR ligand engagementand is known to mediate inflammatory responses to many PAMPs (Akira etal., 2003, Biochem. Soc. Trans. 31:637). The absence of MyD88 in APCsmakes them completely unresponsive to CpG DNA and is therefore thoughtto be essential in all TLR-9-mediated responses (Schnare et al., 2000,Curr. Biol. 10:1139). In contrast, deficiency in MyD88 APCs partiallyeliminates TLR-3-mediated cytokine synthesis but leaves NF-κB, MAPK, andDC maturation responses intact (Alexopoulou et al., 1997, Nature413:732). Therefore to examine the role of MyD88 in TLR ligand-mediatedsurvival responses in activated CD4⁺ T cells, MyD88^(−/−) activated CD4⁺T cells were treated with CpG DNA and poly(I:C) and assessed survival(FIG. 4B). It was observed that MyD88 was required to mediate CpG DNAaugmented survival of activated CD4⁺ T cells. In contrast,poly(I:C)-enhanced survival responses were left intact in MyD88^(−/−)activated CD4⁺ T cells. Therefore, the results presented hereindemonstrate that at least two signaling pathways, MyD88 dependent andMyD88 independent, are capable of mediating direct TLR ligand augmentedsurvival in activated CD4⁺ T cells.

Poly(I:C) or CpG DNA Treatment of Activated CD4⁺ T Cells Up-RegulatesBcl-x_(L) but not Bcl-2 or Bcl-3

Members of the Bcl family are mediators of activated CD4⁺ T cellsurvival. Bcl-2 and BCl-x_(L) are both up-regulated in CD4⁺ T cellsfollowing antigen priming (Boise et al., 1995, Curr. Opin. Immunol.7:620). Bcl-3 has been reported to be up-regulated in activated CD4⁺ Tcells isolated from adjuvant-treated mice and in overexpression studieshas been reported to increase survival (Mitchell et al., 2001, Nat.Immunol. 2:397). Therefore, levels of each of these molecules weremeasured following TLR ligand treatment of activated CD4⁺ T cells (FIGS.4C and 4D). Bcl-2 protein levels were not changed by TLR ligandtreatment relative to untreated activated CD4⁺ T cell controls.Additionally, Bcl-3 protein levels were also left unaffected despite thefact that all of the TLR ligands used in the experiment are also used asadjuvants (Mitchell et al., 2001, Nat. Immunol. 2:397). However,significant increases in BCl-x_(L) protein in CpG DNA and poly(I:C)—treated activated CD4⁺ T cells was observed over LPS-treated anduntreated activated CD4⁺ T cells. Thus, directly mediated activated CD4⁺T cell survival is associated with specific BCl-x_(L) up-regulation.

Poly(I:C) or CpG DNA Treatment of Activated CD4⁺ T Cells Enhances theirSurvival In Vivo

Although TLR ligand-mediated survival of activated CD4⁺ T cells in vitrocould be induced, it still remained uncertain whether these cells had apreferential survival advantage in vivo. To address this question,DO11.10 CD4⁺ T cells were first activated with pOVA-pulsed APCs,purified by magnetic beads, treated with TLR ligands for 16 hours,washed, and then adoptively transferred into congenic BALB/c hosts.Survival and ex vivo proliferative responses were assessed 30 days later(FIGS. 5A and 5B). Consistent with what has been previously reported foractivated effector CD4⁺ T cells, adoptively transferred activatedDO11.10 CD4⁺ T cells seem to preferentially home to the spleen ratherthan to the peripheral lymph nodes (Bradley et al., 1994, J. Exp. Med.180:2401). Importantly, poly(I:C) or CpG DNA treatment of activatedDO11.10 CD4⁺ T cells increased the percentage of recovered T cells inthe host spleen by nearly 2-fold in comparison to spleens from mice thatreceived either LPS-treated or untreated DO11.10 CD4⁺ T cells. Likewise,this 2-fold increase in the percentage of splenic DO11.10 CD4⁺ T cellsfrom hosts that received either poly(I:C)— or CpG DNA-treated activatedDO11.10 CD4⁺ T cells was mirrored by a 2-fold increase in theproliferative response to pOVA in comparison to pOVA-inducedproliferative responses of splenic CD4⁺ T cells from hosts that receivedeither LPS-treated or untreated activated DO11.10 CD4⁺ T cells. Thesedata suggest that TLR ligands improve recall responses by increasing thenumber of antigen-specific CD4⁺ T cells in vivo following activation.

TLR message levels in naive CD44^(low)CD25⁻CD4⁺ T cells and activatedCD4⁺ T cells was first examined. It was found that activated CD4⁺ Tcells, in contrast to naive CD4⁺ T cells, do not express TLR-4 and TLR-2and increase the expression of TLR-3 and TLR-9 in response to TCRengagement. RNA message levels were used as a proxy for expression dueto a lack of antibodies that recognize mouse TLRs. Recent studies havepresented an incomplete picture regarding TCR expression in T cells.Nevertheless, both TLR-3 and TLR-9 messages have been found in restingCD4⁺ T cell preparations where naive and activated cells have not beenfractionated and in mouse T cell lines (Applequist et al., 2002, Int.Immunol. 14:1065; Zarember et al., 2002, J. Immunol. 168:554; Hornung etal., 2002, J. Immunol. 168:4531). In a single report where the TLR-4message was specifically assessed in plate-bound anti-CD3 plus anti-CD28monoclonal antibody-activated regulatory and nonregulatory CD4⁺ T cells,TLR-4 expression was detected in the regulatory but not in thenonregulatory population (Caramalho et al., 2003, J. Exp. Med. 197:403).However, in contrast to the results presented herein and previousstudies, TLR-3 and TLR-9 expression was not found on naive CD4⁺ T cellsalthough activated CD4⁺ T cells were not specifically investigated.

In view of the fact that previous studies conducted with TLRligand-treated APCs from TLR knockout mice demonstrated that NF-κB andMAPK induction requires the expression of cognate TLRs, TLR ligands tovalidate the observed pattern of TLR expression in activated CD4⁺ Tcells (Hemmi et al., 2000, Nature 408:740; Alexopoulou et al., 2001,Nature 413:732; Hoshino et al., 1999, J. Immunol. 162:3749). Poly(I:C)and CpG DNA, but not LPS, induced phosphorylation of I-κB, p38 MAPK,ERK1/2, and JNK/SAPK. Thus, TLR-associated downstream activationpathways are activated by TLR ligands in a manner that matches theobserved pattern of TLR expression in activated CD4⁺ T cells.

To further investigate the possible involvement of TLRs in theseresponses, activated CD4⁺ T cells from mice that are MyD88 deficientwere used. The observed requirement for MyD88 to promote CpGDNA-mediated survival in activated CD4⁺ T cells strongly indicates thatTLR-9 is mediating these responses since all functional responsesmediated by TLR-9 have been reported to be MyD88 dependent (Schnare etal., 2000, Curr. Biol. 10:1139). In contrast, most TLR-3-mediatedpoly(I:C) responses are MyD88 independent including NF-κB activation(Alexopoulou et al., 2001, Nature 413:732). Poly(I:C) can also directlyactivate two intracellular pattern recognition receptors,dsRNA-dependent protein kinase (PKR) and 2′-oligoadenylatesynthetase/RNase L (Diaz-Guerra et al., 1997, Virology 236:354; Gil etal., 1999, Mol. Cell. Biol. 19:4653). Both of these factors whenfunctioning coordinately in virally infected cells inhibit proteintranslation leading to apoptosis, thus making them unlikely targets tomediate poly(I:C)-induced survival. However, intracellular PKRactivation does induce NF-κB and MAPK responses, which provides thepossibility that survival responses may be initiated by this manner(Iordanov et al., 2001, Mol. Cell. Biol. 21:61). The results presentedherein argue that this is quite unlikely since intracellular poly(I:C)PKR-mediated TLR-3-independent responses requires liposomalencapsulation of poly(I:C) (Diebold et al., 2003, Nature 424:324). IfPKR activation does play a role in survival, it may be via an indirectmechanism through its recruitment to the TLR-3 proximal signalingcomplex following poly(I:C) stimulation (Jiang et al., 2003, J. Biol.Chem. 278:16713).

Since TLR ligand-mediated survival in several cell types is NF-κBdependent, it was assessed whether the same were true in activated CD4⁺T cells. It was chosen to inhibit NF-κB activation with NBD, a peptidethat prevents IκB phosphorylation through selectively preventing theassociation of IKKαβ with its regulatory protein NEMO. In theMyD88-dependent TLR signaling pathway, IKKαβ activation has been shownto be requisite for IκB phosphorylation (Wang et al., 2001, Infect.Immun. 69:2270). Moreover, LPS-mediated B cell survival requires thepresence of IKKβ and IKKα (Li et al., 2003, J. Immunol. 170:4630; Kaishoet al., 2001, J. Exp. Med. 193:417). For some functional responses,MyD88-independent IκB phosphorylation may be controlled by two other IKKhomologues, IKKε and TANK-binding kinase 1 (TBK-1), both of which havebeen shown to control poly(I:C)-induced IFN-β synthesis (Tojima et al.,2000, Nature 404:778; Fitzgerald et al., 2003, Nat. Immunol. 4:491). Theobservations of MyD88-independent poly(I:C)-mediated survival inactivated CD4⁺ T cells raises the question of whether IKKκ and TBK-1could also be playing a role in mediating survival responses. Withoutwishing to be bound by any particular theory, it is believed this is notlikely, since NBD which blocks IKKαβ, but not IKKε/TBK-1 activity, wasable to inhibit poly(I:C)-mediated survival of activated CD4⁺ T cells.Moreover, poly(I:C) signaling through TLR-3 has been previously reportedto activate IKKαβ, and catalytically inactive IKKβ mutants inhibitpoly(I:C)-mediated NF-κB-dependent transcription (Gil et al., 2001,Oncogene 20:385; Mitchell et al., 2002, Ann. NY Acad. Sci. 975:114).Thus, the data in activated CD4⁺ T cells indicates that the IKKαβ/NEMOcomplex promotes TLR ligand-mediated NF-κB activation to enhancesurvival. In contrast, the results herein present evidence that MAPK p38or ERK 1/2 activation is not necessary for TLR ligand-activated CD4⁺ Tcell survival.

The effects of TLR ligands on the expression levels of prosurvivalmolecules was also examined. Recognizing studies that suggest thatPAMP-mediated survival may be dependent on Bcl-3 levels (Mitchell etal., 2001, Nat. Immunol. 2:397), levels of this molecule in TLRligand-treated activated CD4⁺ T cells was first assessed. Significantdifferences in Bcl-3 expression in poly(I:C) or CpG DNA-treatedactivated CD4⁺ T cells was not observed when compared with untreatedactivated CD4⁺ T cell controls. These observations may be explained bythe dependence on CD40 costimulation to promote Bcl-3 up-regulation inactivated CD4⁺ T cells in these previous studies (Mitchell et al., 2002,Ann. NY Acad. Sci. 975:114). Bcl-2 levels and BCl-x_(L) levels were alsoexamined in TLR ligand-treated activated CD4⁺ T cells and it was foundthat BCl-x_(L) but not Bcl-2 is up-regulated following TLR ligandtreatment. This result is in agreement with previous work onPAMP-stimulated B cells and DCs (Lundqvist et al., 2002, Cancer Immunol.Immunother. 51:139; Grillot et al., 1996, J. Exp. Med. 183:381). Withoutwishing to be bound by any particular theory, since the BCl-x_(L) geneis known to be a downstream target of NF-κB, it is believed thatBCl-x_(L) is promoting TLR ligand-mediated survival (Caamano et al.,2002, Clin. Microbiol. Rev. 15:414).

In conclusion, the results presented herein provide evidence that TLRligands directly enhance the survival of activated CD4⁺ T cells.TLR-mediated survival had the net effect of increasing expansion andslowing contraction rates of activated CD4⁺ T cells without accentuatingproliferation. It has been hypothesized that adjuvant-induced activatedCD4⁺ T survival can be mediated through APCs by the secretion ofproinflammatory cytokines (Hildeman et al., 2002, Curr. Opin. Immunol.14:354). Interestingly, the results presented herein indicate that fortwo such PAMPs that are effective adjuvants, poly(I:C) and CpG DNA,adjuvant-mediated survival of activated CD4⁺ T cells may not requireAPCs. Moreover, in contrast to the indirect means by which TLR ligandscontrol CD4⁺ T cell responses through APCs, direct effects may allowantigen-specific CD4⁺ T cells to respond to PAMPs in situations whereAPC function is suboptimal, perhaps due to infection (Arrode et al.,2003, Curr. Top. Microbiol. Immunol. 276:277). For example, some viruseswhich use dsRNA intermediates in their own life cycle, also encodeproducts that inhibit DC maturation and cytokine synthesis and therebypromote infection by attenuating appropriate CD4⁺ T cell responses (Judeet al., 2003, Nat. Immunol. 4:573; Engelmayer et al., 1999, J. Immunol.163:6762). This effect could be counteracted by augmented CD4⁺ T cellsurvival responses driven by the release of PAMPs such as dsRNA. Thus,like cells in the innate immune system, activated CD4⁺ T cells may alsoretain the capability to sense the inflammatory environment by directlyresponding to PAMPs. This may represent a novel mechanism by which PAMPspromote adaptive immune responses.

Example 2 Effects of TLR Ligation at the Time of T Cell Stimulation

Mammalian TLRs are a highly conserved family of molecules which havebeen known to have key functions in the innate immune system. There areat present eleven known TLRs. Their extracellular domains bind what havebeen termed PAMPs such as LPS, double stranded RNA, flagellin, CpG DNA,and the like. PAMPs have three key features—they are found only onpathogenic organisms and not on host cells, they are invariant within aclass of organisms, and they are required for pathogen survival (i.e.,escape mutants do not exist).

Until recently, the primary known role for TLRs has been to activatecells of the innate immune system, such as macrophages and dendriticcells (antigen presenting cells—APCs), thus providing an early warningand mechanism of defense until the adaptive immune system (T and Bcells) is able to respond. TLR stimulation of APCs induces theexpression of MHC class II, costimulatory ligands such as CD80 and CD86,and the secretion of inflammatory cytokines such as IL-6, IL-12, IFN-αand IFN-β.

Recently it has been reported that multiple cell types other than innateimmune cells also express TLRs. As discussed elsewhere herein, T cellsexpress TLRs -3, -5, and -9, and ligation of either TLR3 or TLR9 onpre-stimulated T cells induced multiple signaling pathways, includingNF-κB activation. Further, it has been demonstrated that TLR ligationpromoted T cell survival in vitro, an effect which was dependent uponNF-κB. It was observed that augmentation of survival by poly I:C (a TLR3ligand) was MyD88-independent, while augmentation by CpG DNA (a TLR9ligand) was MyD88 dependent. This is consistent with the knownrequirement of MyD88 for TLR9 signaling, and the lack of use of MyD88 byTLR3. FIG. 17A depicts a schematic representation of a signaltransduction pathway involving MyD88.

The present experiments were designed to assess the effects of TLRligation at the time of T cell stimulation, and analysis of signalingpathways which mediate them. As an initial matter, it was demonstratedthat that resting mouse CD4+CD25− (i.e., non-regulatory) T cellsexpressed TLR9 protein, whereas CD4⁺CD25+ Tregs, otherwise known asregulatory T cells, do not (FIG. 6).

It was also observed that TLR9 stimulation of polyclonal T cells (usingCpG DNA, a TLR9 ligand) synergized with sub-mitogenic concentrations ofanti-CD3 monoclonal antibody to induce vigorous proliferation (FIG. 7A).Similar results were observed when anti-CD28 was used in combinationwith anti-CD3 (FIGS. 7B-7D). IL-2 production from treat T cells werealso measured (FIG. 8). These effects were additive to those ofanti-CD28 stimulation.

It was also observed that the effects of CpG DNA and poly I:C did notrequire TRAF6, an adaptor molecule which is believed to couple TLR9 toNF-κB (FIG. 9). This led to the examination of other signaling pathwaysdownstream of TLR9. For example, experiments were designed to assess therole of phosphatidyl inositol 3 kinase (PI3K) in TLR9 activation becauseof the ability of TLR ligation to synergize with TCR stimulation forIL-2 production and proliferation was reminiscent of CD28 functions.PI3K is a lipid kinase which catalyzes the phosphorylation ofphosphatidyl inositol bis 4, 5, phosphate (PIP2) into phosphatidylinositol tris 3, 4, 5, phosphate (PIP3). PIP3 is an important cellsignaling molecule, activating protein kinase B/Akt, and otherdownstream pathways. In T cells, PI3K activation is a major knowndownstream signaling pathway of the T cell costimulatory receptor CD28.

It was observed that CpG DNA activates the PI3K pathway, as indicated bythe expression of phosphorylated Akt (FIG. 10A) and phosphorylated GSK3β(FIG. 10B). CpG-mediated augmentation of IL-2 production andproliferation requires MyD88 and is blocked by PI3K inhibition (FIGS. 11and 12). Sequence analysis shows a potential PI3K binding site in MyD88and therefore experiments can be designed by making appropriate mutantsfor expression in MyD88 deficient T cells to determine the role of thismotif (FIG. 13).

Example 3 In Vivo Effect of TLR Signals on T Cells

To isolate TLR signaling deficiency to T cells, MyD88-deficient mice wasused because MyD88 has been shown to be required for TLR-mediatedeffects, except those through TLR3 and a subset of those through TLR4(MyD88 is also used for the IL-1R and IL-18R). The experimental model isdepicted on FIG. 14. The experimental model is based on the reportedfinding that MyD88-deficient mice die rapidly following T. gondiiinfection, and the ability to make chimeric animals in which the MyD88deficiency is functionally restricted to T cells. Without wishing to bebound by any particular theory, although some of the APCs in thechimeric mice are MyD88 deficient, it is believed they are a smallminority, and the results depicted in FIG. 15 demonstrate that theresponse of APCs in the chimeras to the MyD88-dependent stimulus CpG DNAis essentially normal. Despite these normal APC responses, the datadepicted in Table 1 and FIG. 20 demonstrates that mice lacking MyD88 onT cells are essentially as sensitive to T. gondii as are complete MyD88knockouts. This is associated with decreased early (day 7) serum levelsof both IFN-γ and IL-12 (FIG. 16). The latter in particular issurprising since IL-12 is an APC product and suggests a positivefeedback loop involving IFN-γ and IL-12.

Further, it has been observed that chimeric mice with MyD88-deficient Tcells have similar survival to MyD88−/− mice in that both fail tosurvive the acute phase T. gondii infection.

TABLE 1 (Expected cells Mouse Survival (days) missing MyD88) MyD88−/−mouse 20, 26 all Chimera, TCRα−/− and 31, 61, 62, 62, 63, None MyD88+/+marrow 75+, 75+ Chimera, TCRα−/− and 25, 26, 26, 27, 27, all T cells,MyD88−/− marrow 28, 28 minority of APCs

Experiments were designed to investigate the signaling pathwaysdownstream of TLRs in T cells to determine how activation of PI3K andNF-κB “map” to specific observed effects. Experiments have also beendesigned to determine whether TLR ligation can prevent anergy inductionin T cells. Further, MyD88−/− mice can be crossed onto a TCR transgenicbackground to determine how antigen-specific responses in vivo areinfluenced by TLR signaling pathways on T cells.

Without wishing to be bound by any particular theory, it is believedthat the requirement for MyD88 in the T. gondii system is due to therole of MyD88 in TLR signaling and not in IL-1R or IL-18R signaling.This is based on studies using blocking reagents and/or knockout animalswhich showed no requirement for IL-1 or IL-18 in the response to T.gondii.

Example 4 Expression and Function of TLRs on T Cells

The following experiments were designed to determine the role of MyD88in T cells with respect to the potential PI3K binding site in MyD88following activation of a TLR on T cells. FIG. 17B depicts appropriatemutants for expression in MyD88-deficient T cells to determine the roleof MyD88 with respect to the potential PI3K binding site in MyD88. Itwas observed that optimal IL-6 responses to LPS or IL-1 is dependent onthe Y257 residue in a putative SH2 binding sequence present in the MyD88TIR domain (FIG. 18).

The next set of experiments was designed to assess the effects of CpGODN costimulatory activity in CD4+ T cells. The results demonstrate thatMyD88 is required to activate downstream targets of PI3-kinase such asAkt and GSKα for optimal CpG ODN induced proliferation of CD4+ cells andIL-2 synthesis (FIGS. 19A-19D).

Example 5 Foxp3 Expression in Natural Tregs

The forkhead transcription factor, Foxp3, encoded by the FOXP3 gene, isa marker of regulatory T cells (Tregs). It is known that Foxp3expression, and thus regulatory function, can be induced under certaincircumstances in Foxp3-negative T cells by exposure to TGF-b. It has notbeen established in the art whether Foxp3 expression can be abrogated inpre-existing Foxp3⁺ T cells.

In order to study the effect of stimulation conditions on Foxp3expression, T cells were from mice having a reporter constructintroduced into the FOXP3 locus (Betteli et al., 2006, Nature441:235-238) by sorting cells based on the GFP-reporter construct. Thereporter construct expresses both Fox3p and the fluorescent protein GFP.T cells that are Foxp3⁻ (i.e. GFP⁻) and T cells that are Foxp3⁺ (i.e.GFP⁺) were isolated (FIG. 21A). Foxp3 expression was then assessed underdifferent stimulation conditions,

As shown in FIG. 21B, Foxp3⁺GFP⁺ cells (equivalent to natural Tregs)maintained Foxp3 expression when activated by anti-CD3 and anti-CD28antibodies plus IL-2 or when activated by anti-CD3 plus the TLR3 ligand,poly I:C. However, the TLR9 ligand, CpG, induced a loss of Foxp3expression.

As shown in FIG. 22 (top three panels), and as expected, stimulation ofFoxp3⁻GFP⁻ cells (equivalent to non-regulatory T cells) with anti-CD3and anti-CD28 antibodies and transforming growth factor beta (TGF-b)promoted Foxp3 expression. Unexpectedly, this effect was augmented bythe addition of poly I:C. The effect was not, however, augmented by CpG.The addition of IL-6 to the stimulation conditions blocked the inductionof Foxp3 by TGF-b (FIG. 22, bottom three panels).

Unexpectedly, CpG induced IL-6 production in both stimulated Foxp3⁻ Tcells and stimulated Foxp3⁺ T cells (FIGS. 23A and 23 b). This is thefirst demonstration of induction of IL-6 in T cells by TLR ligands.While not wishing to be bound by theory, it is possible that theinduction of IL-6 by CpG may explain the lack of induction of Foxp3expression by CpG observed in FIG. 21, since IL-6 blocks induction ofFoxp3 expression by TGF-b (FIG. 22).

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A composition for increasing T cell proliferation and cytokineproduction, the composition comprising: a Toll-like receptor (TLR)ligand wherein said TLR ligand is capable of activating a TLR on a Tcell; and a T cell stimulator wherein said stimulator is capable ofactivating said T cell.
 2. The composition of claim 1, wherein said TLRligand is capable of activating TLR9.
 3. The composition of claim 1,wherein said TLR ligand is capable of activating TLR3.
 4. Thecomposition of claim 1, wherein said TLR ligand is selected from thegroup consisting of CpG DNA and poly I:C.
 5. The composition of claim 1,wherein said TLR ligand is a combination of CpG DNA and poly I:C.
 6. Thecomposition of claim 1, wherein said T cell stimulator comprises anantibody selected from the group consisting of an anti-CD3 antibody andan anti-CD28 antibody.
 7. The composition of claim 1, wherein said Tcell stimulator comprises both an anti-CD3 antibody and an anti-CD28antibody.
 8. The composition of claim 1, further comprising an antigenhaving at least one epitope, wherein said epitope is capable ofeliciting an immune response in a mammal.
 9. The composition of claim 1,further comprising a T cell.
 10. The composition of claim 9, whereinsaid T cell is an activated T cell.
 11. The composition of claim 10,wherein said activated T cell exhibits an enhanced survivalcharacteristic.
 12. The composition of claim 1, wherein said cytokine isselected from the group consisting of IL-2 and IL-6.
 13. A compositionfor increasing T cell proliferation and cytokine production, thecomposition comprising a T cell stimulator and one of CpG and poly I:C,wherein said stimulator is capable of activating said T cell.
 14. Thecomposition of claim 13, wherein said T cell stimulator comprises anantibody selected from the group consisting of an anti-CD3 antibody andan anti-CD28 antibody.
 15. The composition of claim 13, wherein said Tcell stimulator comprises both an anti-CD3 antibody and an anti-CD28antibody.
 16. A T cell that is genetically modified to express elevatedlevels TLR3 and/or TLR9 compared to an otherwise identical T cell not somodified, wherein contact of TLR3 and/or TLR9 with a TLR ligand enhancesthe survival of said genetically modified T cell.
 17. The T cell ofclaim 16, wherein said cell exhibits an enhanced survival characteristiccompared to an otherwise identical T cell not so modified.
 18. The cellof claim 16, wherein said cell is capable of regulating an immuneresponse.
 19. The cell of claim 18, wherein said immune response isassociated with a disease selected from the group consisting of aninfectious disease, a cancer, and an autoimmune disease.
 20. A method ofinducing T cell proliferation and promoting cytokine production, themethod comprising activating a T cell with a composition comprising aToll-like receptor (TLR) ligand wherein said TLR ligand is capable ofactivating a TLR on said T cell; and a T cell stimulator wherein saidstimulator is capable of activating said T cell.
 21. The method of claim20, wherein said T cell proliferation is dependent on NF-κB.
 22. Themethod of claim 20, wherein said TLR ligand is capable of activatingTLR9.
 23. The method of claim 20, wherein said TLR ligand is capable ofactivating TLR3.
 24. The method of claim 20, wherein said TLR ligand isselected from the group consisting of CpG DNA and poly I:C.
 25. Themethod of claim 20, wherein said TLR ligand is a combination of CpG DNAand poly I:C.
 26. The method of claim 20, wherein said T cell stimulatorcomprises an antibody selected from the group consisting of an anti-CD3antibody and an anti-CD28 antibody.
 27. The method of claim 20, whereinsaid T cell stimulator comprises both an anti-CD3 antibody and ananti-CD28 antibody.
 28. The method of claim 20, wherein theproliferation of said T cell is independent of the presence of anantigen presenting cell.
 29. The method of claim 20, wherein saidcytokine is selected from the group consisting of IL-2 and IL-6.
 30. Amethod of inducing T cell proliferation and promoting cytokineproduction, the method comprising activating a T cell with a compositioncomprising a T cell stimulator and one of CpG and poly I:C, wherein saidstimulator is capable of activating said T cell.
 31. The method of claim30, wherein said T cell stimulator comprises an antibody selected fromthe group consisting of an anti-CD3 antibody and an anti-CD28 antibody.32. The method of claim 30, wherein said T cell stimulator comprisesboth an anti-CD3 antibody and an anti-CD28 antibody.
 33. The method ofclaim 30, wherein the proliferation of said T cell is independent of thepresence of an antigen presenting cell.
 34. The method of claim 30,wherein said cytokine is selected from the group consisting of IL-2 andIL-6.
 35. A method of enhancing an immune response in a mammal, themethod comprising administering to said mammal a composition comprising:a Toll-like receptor (TLR) ligand wherein said TLR ligand is able toactivate a TLR on said T cell; and a T cell stimulator wherein saidstimulator is able to activate said T cell.
 36. A method of enhancing animmune response in a mammal, the method comprising administering to saidmammal a composition comprising a T cell stimulator and one of CpG andpoly I:C, wherein said stimulator is able to activate said T cell.
 37. Amethod of enhancing an immune response in a mammal, the methodcomprising administering to said mammal a T cell, wherein said T cellhas been stimulated with a composition comprising: a Toll-like receptor(TLR) ligand wherein said TLR ligand is able to activate a TLR on said Tcell; and a T cell stimulator wherein said stimulator is able toactivate said T cell.
 38. A method of enhancing an immune response in amammal, the method comprising administering to said mammal a T cell,wherein said T cell has been stimulated with a composition comprising aT cell stimulator and one of CpG and poly I:C, wherein said stimulatoris able to activate said T cell.
 39. A method of suppressing an immuneresponse in a mammal, the method comprising administering to said mammala composition that inhibits and/or reduces expression of a TLR and/or adownstream signaling molecule thereof in a T cell in said mammal,wherein said composition is selected from the group consisting of asmall interfering RNA (siRNA), an antisense nucleic acid and a ribozyme.40. A method of suppressing an immune response in a mammal, the methodcomprising administering to said mammal a composition that inhibitsand/or reduces activity of a TLR and/or a downstream signaling moleculethereof in a T cell in said mammal, wherein said composition is selectedfrom the group consisting of a transdominant negative mutant, anintracellular antibody, a peptide and a small molecule.
 41. A method formodulating Foxp3 expression in a T cell, the method comprisingactivating a T cell with a composition comprising a T cell stimulatorand one of CpG and poly I:C, wherein said stimulator is capable ofactivating said T cell.
 42. The method of claim 41, wherein thecomposition comprises CpG and Foxp3 expression is reduced.
 43. Themethod of claim 41, wherein the composition comprises poly I:C and Foxp3expression is induced.
 44. The method of claim 43, wherein saidcomposition further comprises transforming growth factor beta (TGF-b).